The purpose of this research is to formulate a molecular mechanism of the action of the sodium pump in cell membranes. The principal goal is to separate the intracellular and extracellular effects of the activating and transported cations and to obtain detailed information on the relationship between cation sites in the partial enzymatic activities and the associated transport modes in human red blood cells. The coupled active transport of Na out of and K into cells is mediated by the plasma membrane Na, K-ATPase. Current ideas on the reaction pathway by high the cardiac qlycoside-sensitive Na, K-ATPase catalyzes the exchange of Na and K are derived from experimental data from two sources, enzymatic studies in systems where all vectorial and transport properties are lost and transport studies in more intact systems, often human erythrocytes, where the precise measurement of enzymatic steps is difficult. In the present work the properties of human erythrocyte ghosts will be utilized where independent control of intracellular and extracellular compartments is possible. Stable, modified ATP or other phosphate-containing substrated can be sealed into ghosts when, following a brief pulse of light, free ATP or substrate is released. Using this new photorelease procedure with caged-ATP or caged-Pi it is now feasible to study the cation requirements for ATP:ADP exchange and for other partial enzymatic activities and the effects of Na, K and their cogeners on enzymatic phosphorylation and dephosphorylation. The cation requirements and effects of various inhibitors on these reactions can then be directly related to the overall enzymatic cycle. The transport modes, Na: Na exchange, K:K exchange and uncoupled Na efflux which are all mediated by the Na pump will be measured in the same system as the partial enzymatic activities. In these studies the way in which intracellular and extracellulartions modulate ATPase activity and ion pumping can be directly related and will facilitate the understanding of this basic transport process at the molecular level.